Methods and Means for Obtaining Modified Phenotypes

ABSTRACT

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells by providing aberrant, preferably unpolyadenylated, target-specific RNA to the nucleus of the host cell. Preferably, the unpolyadenylated, target-specific RNA is provided by transcription of a chimeric gene comprising a promoter and a DNA region encoding the target-specific RNA.

This application is a divisional application of U.S. Ser. No.13/092,645, filed Apr. 22, 2011, now U.S. Pat. No. 9,399,777, issuedJul. 26, 2016, which is a continuation of

U.S. Ser. No. 11/593,056, filed Nov. 6, 2006, now abandoned, which is acontinuation of U.S. Ser. No. 10/152,808, now U.S. Pat. No. 7,138,565,issued Nov. 21, 2006, which is a divisional of U.S. Ser. No. 09/373,720,now U.S. Pat. No. 6,423,885, issued Jul. 23, 2002, the entire contentsof each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to methods for reducing the phenotypic expressionof a nucleic acid of interest in plant cells by providing aberrant RNAmolecules, preferably unpolyadenylated RNA molecules comprising at leastone target specific nucleotide sequence homologous to the nucleic acidof interest, preferably a sense strand, into the nucleus of plant cells.The target-specific unpolyadenylated RNA molecules may be provided byintroduction of chimeric DNAs which when transcribed under control ofconventional promoter and 3′ end formation and polyadenylation regionsyield RNA molecules wherein at least the polyadenylation signal may beremoved by the autocatalytic activity of a self-splicing ribozymecomprised within the transcribed RNA molecules. Also provided are plantcells comprising such RNA molecules or chimeric DNA encoding such RNAmolecules, as well as plants. Similar methods and means for reducing thephenotypic expression of a nucleic acid by co-suppression in eukaryoticcells are provided.

BACKGROUND OF THE INVENTION

Post-transcriptional gene silencing (PTGS) or co-suppression, is acommon phenomenon associated with transgenes in transgenic plants. PTGSresults in sequence-specific removal of the silenced transgene RNA aswell as homologous endogenous gene RNA or viral RNA. It is characterizedby low steady-state mRNA levels with normal (usually high) rates ofnuclear transcription of transgenes being maintained. There are a numberof common features or characteristics for PTGS. PTGS is

-   -   sequence-specific;    -   systemically transmissible;    -   often associated with the presence of multiple copies of        transgenes or with the use of strong promoters;    -   frequently correlated with the presence of repetitive DNA        structures, including inverted repeat T-DNA insertion patterns;    -   often accompanied by de novo DNA methylation in the transcribed    -   region, and    -   may be meiotically reset.

A number of hypothetical models have been proposed to explain PTGS (seee.g. Wassenegger and Pélissier, 1998). Typically, these models suggestthe involvement of a host encoded enzyme

(RNA-directed RNA polymerase (RdRP)) which is proposed to use aberrantRNA as templates to synthesize small copy RNA molecules (cRNA). ThesecRNAs would then hybridize with the target mRNA to form duplexstructures, thereby rendering the mRNA susceptible to degradation byendoribonucleases. So far, there has been no direct evidence that RdRPis involved in PTGS in plants.

An important question arising from the existing models is what type ofRNA is the aberrant RNA that would be used as a template by RdRP, and inwhich cellular compartment RdRP would function.

Several reports have described the accumulation of unproductive orunpolyadenylated transgene RNA in plants which arepost-transcriptionally silenced (Lee et al. 1997; van Eldik et al. 1998;Covey et al., 1997; van Houdt et al., 1997; Metzlaff et al.; 1997).

The following documents relate to methods and means for regulating orinhibiting gene expression in a cell.

U.S. Pat. No. 5,190,131 and EP 0 467 349 A1 describe methods and meansto regulate or inhibit gene expression in a cell by incorporating intoor associating with the genetic material of the cell a non-nativenucleic acid sequence which is transcribed to produce an mRNA which iscomplementary to and capable of binding to the mRNA produced by thegenetic material of that cell.

EP 0 223 399 A1 describes methods to effect useful somatic changes inplants by causing the transcription in the plant cells of negative RNAstrands which are substantially complementary to a target RNA strand.The target RNA strand can be a mRNA transcript created in geneexpression, a viral RNA, or other RNA present in the plant cells. Thenegative RNA strand is complementary to at least a portion of the targetRNA strand to inhibit its activity in vivo.

EP 0 240 208 describes a method to regulate expression of genes encodedfor in plant cell genomes, achieved by integration of a gene under thetranscriptional control of a promoter which is functional in the hostand in which the transcribed strand of DNA is complementary to thestrand of DNA that is transcribed from the endogenous gene(s) one wishesto regulate.

EP 0 647 715 A1 and U.S. Pat. Nos. 5,034,323, 5,231,020 and 5,283,184describe methods and means for producing plants exhibiting desiredphenotypic traits, by selecting transgenotes that comprise a DNA segmentoperably linked to a promoter, wherein transcription products of thesegment are substantially homologous to corresponding transcripts ofendogenous genes, particularly endogenous flavonoid biosynthetic pathwaygenes.

Waterhouse et al. 1998 describe that virus resistance and gene silencingin plants can be induced by simultaneous expression of sense andanti-sense-RNA. The sense and antisense RNA may be located in onetranscript that has self-complementarity.

Hamilton et al. 1998 describes that a transgene with repeated DNA, i.e.inverted copies of its 5′ untranslated region, causes high frequency,posttranscriptional suppression of ACC-oxidase expression in tomato.

WO 98/53083 describes constructs and methods for enhancing theinhibition of a target gene within an organism which involve insertinginto the gene silencing vector an inverted repeat sequence of all orpart of a polynucleotide region within the vector.

WO 95/34688 describes methods for cytoplasmic inhibition of geneexpression and provides genetic constructs for the expression ofinhibitory RNA in the cytoplasm of eukaryotic cells. The inhibitory RNAmay be an anti-sense or a co-suppressor RNA. The genetic constructs arecapable of replicating in the cytoplasm of a eukaryotic cell andcomprise a promoter region, which may be a plant virus subgenomicpromoter in functional combination with the RNA encoding region.

W095/15394 and U.S. Pat. No. 5,908,779 describe a method and constructfor regulating gene expression through inhibition by nuclear antisenseRNA in (mouse) cells. The construct comprises a promoter, antisensesequences, and a cis-or trans-ribozyme which generates 3′-endsindependently of the polyadenylation machinery and thereby inhibits thetransport of the RNA molecule to the cytoplasm.

SUMMARY OF THE INVENTION

The present invention provides a method for reducing the phenotypicexpression of a nucleic acid of interest, which is normally capable ofbeing expressed in a plant cell, the method comprising the step ofproviding to the nucleus of that plant cell aberrant RNA comprising atarget-specific nucleotide sequence, preferably unpolyadenylated RNAcomprising a target specific nucleotide sequence, particularly byproducing aberrant RNA such as unpolyadenylated RNA by transcription ofa chimeric DNA comprised within the plant cell, the chimeric DNAcomprising a plant-expressible promoter operably linked to a targetspecific DNA region encoding that RNA and optionally further comprisinga DNA region involved in 3′ end formation and polyadenylation, precededby a self-splicing ribozyme encoding DNA region.

The invention also provides a method for reducing the phenotypicexpression of a nucleic acid of interest, which is normally capable ofbeing expressed in a plant cell, the method comprising the step ofintroducing into the nuclear genome of the plant cell a chimeric DNA togenerate a transgenic plant cell, the chimeric DNA comprising thefollowing operably linked parts:

-   a plant-expressible promoter region, preferably a constitutive    promoter or an inducible promoter or a tissue-specific promoter;-   a target-specific DNA region encoding a target-specific nucleotide    sequence, preferably a target-specific DNA region comprising a    nucleotide sequence of at least 10 consecutive nucleotides having at    least about 70% sequence identity to about 100% sequence identity to    the nucleic acid of interest or comprising a nucleotide sequence of    at least 10 consecutive nucleotides having at least about 70%    sequence identity to about I00% sequence identity to the complement    of said nucleic acid of interest;-   a DNA region encoding a self-splicing ribozyme, preferably a    self-splicing ribozyme comprising a cDNA copy of a self-splicing    ribozyme from avocado sunblotch viroid, peach latent mosaic viroid,    Chrysanthemum chlorotic mottle viroid, carnation stunt associated    viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley    yellow dwarf virus satellite RNA, arabis mosaic virus satellite RNA,    chicory yellow mottle virus satellite RNA S1, lucerne transient    streak virus satellite RNA, tobacco ringspot virus satellite RNA,    subterranean clover mottle virus satellite RNA, solanum nodiflorum    mottle virus satellite RNA, velvet tobacco mottle virus satellite    RNA, Cherry small circular viroid-like RNA or hepatitis delta virus    RNA, particularly a DNA region comprising the nucleotide sequence of    SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and-   a) a DNA region involved in 3′ end formation and polyadenylation;

wherein said chimeric DNA when transcribed produces a first RNA moleculecomprising a target specific nucleotide sequence and a self-splicingribozyme, which when cleaved by autocatalysis produces a second RNAmolecule comprising a target specific nucleotide sequence wherein the 3′end of the first RNA molecule comprising the polyadenylation site hasbeen removed. Optionally, a transgenic plant may be regenerated from thetransgenic plant cell. Preferably, the DNA region encoding aself-splicing ribozyme is located immediately upstream of the DNA regioninvolved in 3′ end formation and polyadenylation.

It is another objective of the invention to provide a chimeric DNAmolecule for reducing the phenotypic expression of a nucleic acid ofinterest, which is normally capable of being expressed in a plant cell,comprising

-   a plant-expressible promoter region, preferably a constitutive    promoter or an inducible promoter or a tissue-specific promoter;-   a target-specific DNA region encoding a target-specific nucleotide    sequence, preferably a target-specific DNA region comprising a    nucleotide sequence of at least 10 consecutive nucleotides having at    least about 70% sequence identity to about 100% sequence identity to    the nucleic acid of interest or comprising a nucleotide sequence of    at least 10 consecutive nucleotides having at least about 70%    sequence identity to about 100% sequence identity to the complement    of said nucleic acid of interest;-   a DNA region encoding a self-splicing ribozyme, preferably a    self-splicing ribozyme comprising a cDNA copy of a self-splicing    ribozyme from avocado sunblotch viroid, peach latent mosaic viroid,    Chrysanthemum chlorotic mottle viroid, carnation stunt associated    viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley    yellow dwarf virus satellite RNA, arabis mosaic virus satellite RNA,    chicory yellow mottle virus satellite RNA S1, lucerne transient    streak virus satellite RNA, tobacco ringspot virus satellite RNA,    subterranean clover mottle virus satellite RNA, solanum nodiflorum    mottle virus satellite RNA, velvet tobacco mottle virus satellite    RNA, Cherry small circular viroid-like RNA or hepatitis delta virus    RNA, particularly a DNA region comprising the nucleotide sequence of    SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and-   a DNA region involved in 3′ end formation and polyadenylation;

wherein said chimeric DNA when transcribed produces a first RNA moleculecomprising a target specific nucleotide sequence and a self-splicingribozyme, which when cleaved by autocatalysis produces a second RNAmolecule comprising a target specific nucleotide sequence wherein the 3′end of the first RNA molecule comprising the polyadenylation site hasbeen removed. Preferably, the DNA region encoding a self-splicingribozyme is located immediately upstream of the DNA region involved in3′ end formation and polyadenylation.

It is yet another objective of the invention to provide plant cells andplants comprising a nucleic acid of interest which is normally capableof being phenotypically expressed, further comprising a chimeric DNA,preferably stably-integrated into the nuclear genome, comprising

-   a plant-expressible promoter region, preferably a constitutive    promoter or an inducible promoter or a tissue-specific promoter;-   a target-specific DNA region encoding a target-specific nucleotide    sequence, preferably a target-specific DNA region comprising a    nucleotide sequence of at least 10 consecutive nucleotides having at    least about 70% sequence identity to about 100% sequence identity to    the nucleic acid of interest or comprising a nucleotide sequence of    at least 10 consecutive nucleotides having at least about 70%    sequence identity to about 100% sequence identity to the complement    of said nucleic acid of interest;-   a DNA region encoding a self-splicing ribozyme, preferably a    self-splicing ribozyme comprising a cDNA copy of a self-splicing    ribozyme from avocado sunblotch viroid, peach latent mosaic viroid,    Chrysanthemum chlorotic mottle viroid, carnation stunt associated    viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley    yellow dwarf virus satellite RNA, arabis mosaic virus satellite RNA,    chicory yellow mottle virus satellite RNA S1, lucerne transient    streak virus satellite tobacco ringspot virus satellite RNA,    subterranean clover mottle virus satellite RNA, solanum nodiflorum    mottle virus satellite RNA, velvet tobacco mottle virus satellite    RNA, Cherry small circular viroid-like RNA or hepatitis delta virus    RNA, particularly a DNA region comprising the nucleotide sequence of    SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and-   a DNA region involved in 3′ end formation and polyadenylation;

wherein said chimeric DNA when transcribed produces a first RNA moleculecomprising a target specific nucleotide sequence and a self-splicingribozyme, which when cleaved by autocatalysis produces a second RNAmolecule comprising a target specific nucleotide sequence wherein the 3′end of the first RNA molecule comprising the polyadenylation site hasbeen removed.

The invention also provides a method for identifying a phenotypeassociated with the expression of a nucleic acid of interest in a plantcell, the method comprising:

-   1) selecting within the nucleic acid of interest a target sequence    of at least 5 consecutive nucleotides;-   2) introducing a chimeric DNA into the nucleus of a suitable plant    host cell comprising the nucleic acid of interest, the chimeric DNA    comprising the following operably linked DNA fragments:    -   -   a) a plant-expressible promoter region;        -   b) a target-specific DNA region comprising a nucleotide            sequence of at least about 70% to about 100% sequence            identity to said target sequence or to the complement of            said target sequence; followed by        -   c) a DNA region encoding a self-splicing ribozyme located            immediately upstream of        -   d) a DNA region involved in 3′ end formation and            polyadenylation;-   3) observing the phenotype by a suitable method.

Yet another objective of the invention is to provide a method forreducing the phenotypic expression of a nucleic acid of interest, whichis normally capable of being expressed in a eukaryotic cell, the methodcomprising the step of providing to the nucleus of said eukaryotic cellaberrant RNA, preferably unpolyadenylated RNA, comprising a targetspecific nucleotide sequence of at least 10 consecutive nucleotides withat least about 70% sequence identity to about 100% sequence identity tothe nucleotide sequence of the nucleic acid of interest, particularly byproducing aberrant RNA such as unpolyadenylated RNA by transcription ofa chimeric DNA comprised within the eukaryotic cell, the chimeric DNAcomprising a plant-expressible promoter operably linked to a targetspecific DNA region encoding that RNA and optionally further comprisinga DNA region involved in 3′ end formation and polyadenylation, precededby a self-splicing ribozyme encoding DNA region.

Still another objective of the invention is to provide a method forreducing the phenotypic expression of a nucleic acid of interest, whichis normally capable of being expressed in a eukaryotic cell, comprisingthe step of introducing into the nuclear genome of the eukaryotic cell achimeric DNA to generate a transgenic plant cell, comprising thefollowing operably linked parts:

-   -   a) a promoter region functional in the eukaryotic cell;    -   b) a target-specific DNA region comprising nucleotide sequence        of at least 10 consecutive nucleotides with at least about 70%        sequence identity to about 100% sequence identity to the        nucleotide sequence of the nucleic acid of interest;    -   c) a DNA region encoding a self-splicing ribozyme; and    -   d) a DNA region involved in 3′ end formation and polyadenylation

wherein the chimeric DNA when transcribed produces a first RNA moleculecomprising a target specific nucleotide sequence and a self-splicingribozyme, which when cleaved by autocatalysis produces a second RNAmolecule comprising a target specific nucleotide sequence wherein the 3′end of the first RNA molecule comprising the polyadenylation site hasbeen removed.

The invention also provides a eukaryotic cell comprising a nucleic acidof interest, normally capable of being phenotypically expressed, furthercomprising a chimeric DNA comprising the following operably linkedparts:

-   a promoter region functional in the eukaryotic cell;-   a target-specific DNA region comprising nucleotide sequence of at    least 10 consecutive nucleotides with at least about 70% sequence    identity to about 100% sequence identity to the nucleotide sequence    of the nucleic acid of interest;-   a DNA region encoding a self-splicing ribozyme; and-   a DNA region involved in 3′ end formation and polyadenylation    wherein said chimeric DNA when transcribed in the eukaryotic cell    produces a first RNA molecule comprising a target specific    nucleotide sequence and a self-splicing ribozyme, which when cleaved    by autocatalysis produces a second RNA molecule comprising a target    specific nucleotide sequence wherein the 3′ end of the first RNA    molecule comprising the polyadenylation site has been removed, as    well as non-human eukaryotic organisms comprising or consisting    essentially of such eukaryotic cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the ribozyme-containing GUS chimericgene (pMBW267 and pMBW259) the control construct (pMBW 265) and the GUSchimeric gene used for supertransformation (pBPPGH). 35s-P: CaMV 35Spromoter; GUS: region encoding β-glucuronidase; SAT: cDNA copy of thesatellite RNA of Barley Yellow Dwarf Virus (BYDV) in positive strandorientation (−) or in minus strand orientation (−); Ocs-T: region fromthe octopine synthase gene from Agrobacterium involved in 3′ endformation and polyadenylation; 3′ Sat: cDNA copy of the 3′ end of thesatellite RNA of BYDV; 5′ SAT: cDNA copy of the 5′ end of the satelliteRNA of BYDV; PP2-P: 1.3 kb promoter region of a gene encoding thecucurbit phloem protein PP2; Nos-T: region from the nopaline synthasegene from Agrobacterium involved in 3′ end formation andpolyadenylation; C: autocatalytic cleavage site in the RNA moleculetranscribed from the chimeric gene.

FIG. 2A represents schematically the different sense and antisenseconstructs, as well as the so-called CoP (complementary pair) constructsused for reducing the phenotypic expression of a transgenic Gus gene.

FIG. 2B represents schematically the different sense and antisenseconstructs used for obtaining virus resistance.

FIG. 3A represents schematically the so-called panhandle construct orCoP constructs used for reducing the phenotypic expression of a Δ12desaturase gene in Arabidopsis (Nos Pro: nopaline synthase genepromoter; nptII neomycin phospho-transferase coding region; Nos term:nopaline syntase gene terminator; FP1: truncated seed specific napinpromoter; 480 bp: 5′ end of the Fad2 gene of Arabidopsis thaliana insense orientation; 623 bp: spacer; 480 bp: 5′ end of the Fad2 gene ofArabidopsis thaliana in antisense orientation.

FIG. 3B represents schematically a common cosuppression construct forfor reducing the phenotypic expression of a Δ12 desaturase gene inArabidopsis thaliana.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Although gene-silencing, either by anti-sense RNA or throughco-suppression using sense RNA, is a commonly observed phenomenon intransgenic-research, the intentional generation of gene-silencedtransgenic eukaryotic cells and transgenic organisms, particularly plantcells and plants, still faces a number of problems. In particular theefficiency of gene-silencing is still amenable to improvement, both innumber of transgenic lines exhibiting the phenomenon as well as in thelevel of reduction of transcription and ultimately the phenotypicexpression of particular nucleic acid of interest in a particulartransgenic line.

A number of improved methods for gene-silencing have already beendescribed, e.g. the simultaneous use in one cell of anti-sense and senseRNA targeted to the nucleic acid of interest, preferably co-located onone transcript exhibiting self-complementarity. Novel methods forincreasing the efficiency of gene-silencing, preferably gene-silencingthrough co-suppression in a eukaryotic cell or organism, preferablyplant cell or plant, and means therefore, are described in the differentembodiments provided by the specification and claims.

The current invention is based on the unexpected observation by theinventors, that the provision or the introduction of aberranttarget-specific RNA, preferably unpolyadenylated target-specific RNA,particularly an aberrant target-specific

RNA comprising a nucleotide sequence essentially identical to thenucleic acid of interest in sense orientation, into the nucleus of acell of a eukaryotic organism, particularly a cell of plant, resulted inan efficient reduction of the expression of the nucleic acid ofinterest, both in the level of reduction as well as in the number oftransgenic lines exhibiting gene-silencing. The understanding ofhypothetical mechanisms through which gene-silencing, particularly PTGS,is supposed to proceed did not allow to predict that among all variablespotentially involved in initiation and maintenance of gene-silencing,the selection of this one parameter—i.e. providing aberrant, preferablyunpolyadenylated RNA—would have been sufficient to significantlyincrease the efficiency of gene-silencing, particularly gene-silencingthrough co-suppression.

In one embodiment of the invention, a method is provided for reducingthe phenotypic expression of a nucleic acid of interest, which isnormally capable of being expressed in a plant cell, comprising the stepof providing aberrant RNA such as unpolyadenylated RNA which includes atarget-specific nucleotide sequence to the nucleus of that plant cell.Conveniently, the aberrant RNA such as unpolyadenylated RNA includingthe target-specific nucleotide sequence may be produced by transcriptionof a chimeric DNA or chimeric gene comprised within the plant cell,preferably incorporated, particularly stably integrated into the nucleargenome of the plant cell. In a particularly preferred embodiment, theaberrant RNA is unpolyadenylated RNA which, still exhibits othermodifications characteristic of mRNA, such as, but not limited to, thepresence of a cap-structure at the 5′ end.

As used herein, the term “expression of a gene” refers to the processwherein a DNA region which is operably linked to appropriate regulatoryregions, particularly to a promoter region, is transcribed into an RNAwhich is biologically active i.e., which is either capable ofinteraction with another nucleic acid or which is capable of beingtranslated into a polypeptide or protein. A gene is said to encode anRNA when the end product of the expression of the gene is biologicallyactive RNA, such as e.g. an antisense RNA, a ribozyme or a replicativeintermediate. A gene is said to encode a protein when the end product ofthe expression of the gene is a protein or polypeptide.

A nucleic acid of interest is “capable of being expressed”, when saidnucleic acid, when introduced in a suitable host cell, particularly in aplant cell, can be transcribed (or replicated) to yield an RNA, and/ortranslated to yield a polypeptide or protein in that host cell.

The term “gene” means any DNA fragment comprising a DNA region (the“transcribed DNA region”) that is transcribed into a RNA molecule (e.g.,a mRNA) in a cell operably linked to suitable regulatory regions, e.g.,a plant-expressible promoter. A gene may thus comprise several operablylinked DNA fragments such as a promoter, a 5′ leader sequence, a codingregion, and a 3′ region comprising a polyadenylation site. A plant geneendogenous to a particular plant species (endogenous plant gene) is agene which is naturally found in that plant species or which can beintroduced in that plant species by conventional breeding. A chimericgene is any gene which is not normally found in a plant species or,alternatively, any gene in which the promoter is not associated innature with part or all of the transcribed DNA region or with at leastone other regulatory region of the gene.

As used herein, “phenotypic expression of a nucleic acid of interest”refers to any quantitative trait associated with the molecularexpression of a nucleic acid in a host cell and may thus include thequantity of RNA molecules transcribed or replicated, the quantity ofpost-transcriptionally modified RNA molecules, the quantity oftranslated peptides or proteins, the activity of such peptides orproteins.

A “phenotypic trait” associated with the phenotypic expression of anucleic acid of interest refers to any quantitative or qualitativetrait, including the trait mentioned, as well as the direct or indirecteffect mediated upon the cell, or the organism containing that cell, bythe presence of the RNA molecules, peptide or protein, orposttranslationally modified peptide or protein. The mere presence of anucleic acid in a host cell, is not considered a phenotypic expressionor a phenotypic trait of that nucleic acid, even though it can bequantitatively or qualitatively traced. Examples of direct or indirecteffects mediated on cells or organisms are, e.g., agronomically orindustrial useful traits, such as resistance to a pest or disease;higher or modified oil content etc.

As used herein, “reduction of phenotypic expression” refers to thecomparison of the phenotypic expression of the nucleic acid of interestto the eukaryotic cell in the presence of the RNA or chimeric genes ofthe invention, to the phenotypic expression of the nucleic acid ofinterest in the absence of the RNA 10 or chimeric genes of theinvention. The phenotypic expression in the presence of the chimeric RNAof the invention should thus be lower than the phenotypic expression inabsence thereof, preferably be only about 25%, particularly only about10%, more particularly only about 5% of the phenotypic expression inabsence of the chimeric RNA, especially the phenotypic expression shouldbe completely inhibited for all practical purposes by the presence ofthe chimeric RNA or the chimeric gene encoding such an RNA.

A reduction of phenotypic expression of a nucleic acid where thephenotype is a qualitative trait means that in the presence of thechimeric RNA or gene of the invention, the phenotypic trait switches toa different discrete state when compared to a situation in which suchRNA or gene is absent. A reduction of phenotypic expression of a nucleicacid may thus, a.o., be measured as a reduction in transcription of(part of) that nucleic acid, a reduction in translation of (part of)that nucleic acid or a reduction in the effect the presence of thetranscribed RNA(s) or translated polypeptide(s) have on the eukaryoticcell or the organism, and will ultimately lead to altered phenotypictraits. It is clear that the reduction in phenotypic expression of anucleic acid of interest, may be accompanied by or correlated to anincrease in a phenotypic trait.

As used herein “a nucleic acid of interest” or a “target nucleic acid”refers to any particular RNA molecule or DNA sequence which may bepresent in a eukaryotic cell, particularly a plant cell.

As used herein “aberrant RNA” refers to polyribonucleotide moleculeswhich have characteristic differing from mRNA molecules normally foundin that cell. The different characteristics include but are not limitedto the absence or removal of a 5′ cap structure, presence of persistentintrons e.g. introns which have been modified in their splice sites soas to prevent splicing, or the absence of the polyA tail normally foundassociated with the mRNA (“unpolyadenylated RNA”).

The term “target-specific nucleotide sequence” as used herein, refers toa nucleotide sequence (either DNA or RNA nucleotide sequence dependingon the context) which can reduce the expression of the target nucleicacid of interest by gene-silencing. Preferably, only the expression ofthe target nucleic acid or gene, or nucleic acids or genes comprisingessentially similar nucleotide sequence is reduced.

Preferably the target-specific nucleotide sequence comprises anucleotide sequence corresponding to the “sense” nucleotide sequence ofthe nucleic acid or gene of interest. In other words, a target-specificsense nucleotide sequence may be essentially similar to part of an RNAmolecule transcribed or produced from the nucleic acid or gene ofinterest or to parts of the nucleic acid or gene of interest controllingthe production of that transcribed or produced RNA molecule, when readin the same 5′ to 3′ direction as the transcribed or produced RNAmolecule.

Preferably, the target specific nucleotide sequence corresponds to partof a nucleic acid region from which RNA is produced, particularly aregion which is transcribed and translated. It is particularly preferredthat the target sequence corresponds to one or more consecutive exons,more particularly is located within a single exon of a coding region.However, the target specific nucleotide sequence may also becorresponding to untranslated regions of the RNA molecule produced fromthe nucleic acid or gene of interest. Moreover, in the light of a recentpublication by Mette et al. (1999), it is expected that the targetspecific nucleotide sequence may also correspond to the regionscontrolling the production or transcription of RNA from the nucleotideor gene of interest, such as the promoter region.

The length of the sense target-specific nucleotide sequence may varyfrom about 10 nucleotides (nt) up to a length equaling the length (innucleotides) of the target nucleic acid. Preferably the total length ofthe sense nucleotide sequence is at least 10 nt, preferably 15 nt,particularly at least about 50 nt, more particularly at least about 100nt, especially at least about 150 nt, more especially at least about 200nt, quite especially at least about 550 nt. It is expected that there isno upper limit to the total length of the sense nucleotide sequence,other than the total length of the target nucleic acid. However forpractical reason (such as e.g. stability of the chimeric genes) it isexpected that the length of the sense nucleotide sequence should notexceed 5000 nt, particularly should not exceed 2500 nt and could belimited to about 1000 nt.

It will be appreciated that the longer the total length of the sensenucleotide sequence is, the less stringent the requirements for sequenceidentity between the total sense nucleotide sequence and thecorresponding sequence in the target nucleic acid or gene become.Preferably, the total sense nucleotide sequence should have a sequenceidentity of at least about 75% with the corresponding target sequence,particularly at least about 80%, more particularly at least about 85%,quite particularly about 90%, especially about 95%, more especiallyabout 100%, quite especially be identical to the corresponding part ofthe target nucleic acid. However, it is preferred that the sensenucleotide sequence always includes a sequence of about 10 consecutivenucleotides, particularly about 20 nt, more particularly about 50 nt,especially about 100 nt, quite especially about 150 nt with 100%sequence identity to the corresponding part of the target nucleic acid.Preferably, for calculating the sequence identity and designing thecorresponding sense sequence, the number of gaps should be minimized,particularly for the shorter sense sequences.

As used herein, “sequence identity” with regard to nucleotide sequences(DNA or RNA), refers to the number of positions with identicalnucleotides divided by the number of nucleotides in the shorter of thetwo sequences. The alignment of the two nucleotide sequences isperformed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983)using a window-size of 20 nucleotides, a word length of 4 nucleotides,and a gap penalty of 4. Computer-assisted analysis and interpretation ofsequence data, including sequence alignment as described above, can,e.g., be conveniently performed using the programs of theIntelligeneticsm Suite (Intelligenetics Inc., CA). Sequences areindicated as “essentially similar” when such sequence have a sequenceidentity of at least about 75%, particularly at least about 80%, moreparticularly at least about 85%, quite particularly about 90%,especially about 95%, more especially about 100%, quite especially areidentical. It is clear than when RNA sequences are said to beessentially similar or have a certain degree of sequence identity withDNA sequences, thymine (T) in the DNA sequence is considered equal touracil (U) in the RNA sequence.

It is expected however, that the target-specific nucleotide sequence mayalso comprise a nucleotide sequence corresponding to the “antisense”nucleotide sequence of the nucleic acid or gene of interest. In otherwords, a target-specific antisense nucleotide sequence may beessentially similar to the complement of part of an RNA moleculetranscribed or produced from the nucleic acid or gene of interest or tothe complement of parts of the nucleic acid or gene of interestcontrolling the production of that transcribed or produced RNA molecule,when read in the same 5′ to 3′ direction as the transcribed or producedRNA molecule.

The requirements for antisense target-specific nucleotide sequences withregard to length, similarity etc. are expected to be essentially similaras for sense target-specific nucleotide sequences as specified herein.

It will be clear to the person skilled in the art that theunpolyadenylated RNA molecule may comprise more than one target-specificnucleotide sequence and particularly that the unpolyadenylated RNAmolecule may comprise sense and antisense target-specific nucleotidesequences wherein the sense and antisense nucleotide sequences areessentially complementary to each other and capable of forming anartificial hairpin structure as described in Waterhouse et at., 1998 orin PCT-application PCT/IB99/00606 (incorporated by reference). The RNAmolecule may comprise a spacer nucleotide sequence located between thesense and antisense nucleotide sequence. It is expected that there areno length limits or sequence requirements associated with the spacerregion, as long as these parameters do not interfere with the capabilityof the RNA regions with the sense and antisense nucleotide sequence toform a double stranded RNA. In a preferred embodiment, the spacer regionvaries in length from 4 to about 200 bp.

“Hairpin RNA” refers to any self-annealing double stranded RNA molecule.In its simplest representation, a hairpin RNA consists of a doublestranded stem made up by the annealing RNA strands, connected by asingle stranded RNA loop, and is also referred to as a “pan-handle RNA.”

Thus, it will be clear to the person skilled in the art that theconstructs of examples 4-8 hereinbelow which produce target-specifichairpin RNA may be modified to produce unpolyadenylated target-specifichairpin RNA when transcribed. Provision of unpolyadenylatedtarget-specific hairpin RNA into the nucleus of a cell of a eukaryoticorganism would result in an efficient reduction of the expression of thenucleic acid of interest, both in the level of reduction as well as inthe number of transgenic lines exhibiting gene-silencing. Providingaberrant, preferably unpolyadenylated hairpin RNA would be sufficient tosignificantly increase the efficiency of gene-silencing.

As indicated above, introduction of target-specific unpolyadenylated RNAinto the nucleus of a plant cell can conveniently be achieved bytranscription of a chimeric DNA encoding RNA introduced into thenucleus, preferably stably integrated into the nuclear genome of a plantcell.

In a preferred embodiment of the invention, the target-specificunpolyadenylated RNA may be produced in the nucleus of a plant cell bytranscription of a chimeric DNA encoding a first target-specific RNA,which may be further processed by the action of a ribozyme also present,and preferably also encoded by a chimeric gene, in the plant cell toyield a second unpolyadenylated target-specific RNA. It will be clearfor the person skilled in the art that the RNA processing need not besubsequently but can occur simultaneously. In a particularly preferredembodiment the ribozyme is a self-splicing ribozyme which is comprisedwithin the generated target specific RNA transcript.

Thus, in a particularly preferred embodiment of the invention, a methodis provided for reducing the phenotypic expression of a nucleic acid ofinterest, which is normally capable of being expressed in a plant cell,the method comprising the step of introducing into the nuclear genome ofthe plant cell a chimeric DNA to generate a transgenic plant cell, thechimeric DNA comprising the following operably linked parts:

-   -   (a) a plant-expressible promoter region;    -   (b) a target-specific DNA region;    -   (c) a DNA region encoding a self-splicing ribozyme; and    -   (d) a DNA region involved in 3′ end formation and        polyadenylation        -   wherein the chimeric DNA when transcribed produces a first            RNA molecule comprising a target specific nucleotide            sequence and a self-splicing ribozyme, which when cleaved by            autocatalysis produces a second RNA molecule comprising a            target specific nucleotide sequence wherein the 3′ end of            the first RNA molecule comprising the polyadenylation site            has been removed.

The method may optionally further comprise the step of regenerating athe transgenic plant cell into a transgenic plant.

As used herein, “a ribozyme” is a catalytic RNA molecule that has theintrinsic ability to break and form covalent bonds in ribonucleic acidsat specific sites in the absence of a cofactor other than a divalentcation.

As used herein a “self-splicing ribozyme” or “self-cleaving ribozyme” isa ribozyme capable of autocatalysis at a specific site within thatribozyme. Preferred self-splicing ribozymes are self-splicing ribozymeswith a so-called hammerhead structure. However, it is expected thatself-cleaving ribozymes with another conformation such as the hairpinself-cleaving structures encountered in the minus strand of replicationintermediates of e.g. the nepoviruses can also be used to the sameeffect.

Particularly preferred self-splicing ribozymes are those involved in thereplication of small circular plant pathogenic RNAs, such as but notlimited to the self-splicing ribozyme from avocado sunblotch viroid,peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid,carnation stunt associated viroid, Newt satellite 2 transcript,Neurospora VS RNA, barley yellow dwarf virus satellite RNA, arabismosaic virus satellite RNA, chicory yellow mottle virus satellite RNAS1, lucerne transient streak virus satellite RNA, tobacco ringspot virussatellite RNA, subterranean clover mottle virus satellite RNA, solanumnodiflorum mottle virus satellite RNA, velvet tobacco mottle virussatellite RNAvSCMoV or Cherry small circular viroid-like RNAcscRNA1.Table 1 lists different variant ribozymes suitable for the invention, aswell as a reference to their nucleotide sequence.

The DNA regions encoding self-splicing ribozymes may be cDNA copies ofpart of the mentioned plant pathogenic RNAs comprising the ribozyme, ormay be synthetic DNA. Also comprised are variants such as mutantsincluding substitutions, deletions or insertions of nucleotides withinthe ribozyme nucleotide sequence in such a way that the autocatalyticcapacity of the ribozymes is not substantially altered.

Preferably, the DNA region encoding the self-splicing ribozyme islocated immediately upstream of the DNA region encoding the 3′ endformation and polyadenylation signal. However, having read thespecification, the person skilled in the art will immediately realizethat the DNA region encoding the self-splicing ribozyme may be comprisedwithin the chimeric gene encoding the unpolyadenylated RNA at otherlocations, provided that a sufficiently large second RNA comprising atarget-specific nucleotide wherein the polyadenylation site is removedmay be generated.

TABLE 1 Different self-cleaving ribozymes Accession RNA SpeciesReference Nr (+) strand (−) strand Avocado sunblotch viroid Symons 1981Nucleic Acids Res. JO2020 hammerhead hammerhead 9 6527-6537 Avocadosunblotch viroid variant C-10 Rakowski 1989 Virology 173 352-356 M31100hammerhead hammerhead and Symons Avocado sunblotch viroid variant B-1Rakowski 1989 Virology 173 352-356 M31086 hammerhead hammerhead andSymons Avocado sunblotch viroid variant A-2 Rakowski 1989 Virology 173352-356 M31085 hammerhead hammerhead and Symons Avocado sunblotch viroidvariant B-2 Rakowski 1989 Virology 173 352-356 M31087 hammerheadhammerhead and Symons Avocado sunblotch viroid variant C-2 Rakowski 1989Virology 173 352-356 M31092 hammerhead hammerhead and Symons Avocadosunblotch viroid variant B-3 Rakowski 1989 Virology 173 352-356 M31088hammerhead hammerhead and Symons Avocado sunblotch viroid variant C-3Rakowski 1989 Virology 173 352-356 M31093 hammerhead hammerhead andSymons Avocado sunblotch viroid variant B-4 Rakowski 1989 Virology 173352-356 M31089 hammerhead hammerhead and Symons Avocado sunblotch viroidvariant C-4 Rakowski 1989 Virology 173 352-356 M31094 hammerheadhammerhead and Symons Avocado sunblotch viroid variant B-5 Rakowski 1989Virology 173 352-356 M31090 hammerhead hammerhead and Symons Avocadosunblotch viroid variant C-5 Rakowski 1989 Virology 173 352-356 M31095hammerhead hammerhead and Symons Avocado sunblotch viroid variant B-6Rakowski 1989 Virology 173 352-356 M31091 hammerhead hammerhead andSymons Avocado sunblotch viroid variant C-6 Rakowski 1989 Virology 173352-356 M31096 hammerhead hammerhead and Symons Avocado sunblotch viroidvariant C-7 Rakowski 1989 Virology 173 352-356 M31097 hammerheadhammerhead and Symons Avocado sunblotch viroid variant C-8 Rakowski 1989Virology 173 352-356 M31098 hammerhead hammerhead and Symons Avocadosunblotch viroid variant C-9 Rakowski 1989 Virology 173 352-356 M31099hammerhead hammerhead and Symons Avocado sunblotch viroid ASBVd-BSemancik and 1994 J. Gen Virol. 75 1543-1549 S74687 hammerheadhammerhead Szychowski Avocado sunblotch viroid ASBVd-V Semancik and 1994J. Gen Virol. 75 1543-1549 S73861 hammerhead hammerhead Szychowski Peachlatent mosaic viroid PLMVd.1 Hernandez 1992 Proc. Natl. Acad. Sci.M83545 hammerhead hammerhead and Flores 89 3711-3715 Peach latent mosaicviroid PLMVd.2 Hernandez 1992 Proc. Natl. Acad. Sci. hammerheadhammerhead and Flores 89 3711-3715 Peach latent mosaic viroidPeach-Italy Schamloul et al. 1995 Acta Hortic. 386 522-530 hammerheadhammerhead Peach latent mosaic viroid Cherry-Canada Hadini et al. 1997Plant Dis. 81, 154-158 hammerhead hammerhead Peach latent mosaic viroidvariant gds2 Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005294hammerhead hammerhead Peach latent mosaic viroid variant gds21 Ambros etal. 1998 J. Virol. 72 7397-7406 AJ005295 hammerhead hammerhead Peachlatent mosaic viroid variant gds15 Ambros et al. 1998 J. Virol. 727397-7406 AJ005296 hammerhead hammerhead Peach latent mosaic viroidvariant gds23 Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005297hammerhead hammerhead Peach latent mosaic viroid variant gds18 Ambros etal. 1998 J. Virol. 72 7397-7406 AJ005298 hammerhead hammerhead Peachlatent mosaic viroid variant gds1 Ambros et al. 1998 J. Virol. 727397-7406 AJ005299 hammerhead hammerhead Peach latent mosaic viroidvariant gds3 Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005300hammerhead hammerhead Peach latent mosaic viroid variant gds19 Ambros etal. 1998 J. Virol. 72 7397-7406 AJ005301 hammerhead hammerhead Peachlatent mosaic viroid variant gds13 Ambros et al. 1998 J. Virol. 727397-7406 AJ005302 hammerhead hammerhead Peach latent mosaic viroidvariant gds6 Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005303hammerhead hammerhead Peach latent mosaic viroid variant gds16 Ambros etal. 1998 J. Virol. 72 7397-7406 AJ005304 hammerhead hammerhead Peachlatent mosaic viroid variant esc8 Ambros et al. 1998 J. Virol. 727397-7406 AJ005305 hammerhead hammerhead Peach latent mosaic viroidvariant esc16 Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005306hammerhead hammerhead Peach latent mosaic viroid variant esc5 Ambros etal. 1998 J, Virol. 72 7397-7406 AJ005307 hammerhead hammerhead Peachlatent mosaic viroid variant esc12 Ambros et al. 1998 J. Virol. 727397-7406 AJ005308 hammerhead hammerhead Peach latent mosaic viroidvariant esc 10 Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005309hammerhead hammerhead Peach latent mosaic viroid variant esc 14 Ambroset al. 1998 J. Virol. 72 7397-7406 AJ005310 hammerhead hammerhead Peachlatent mosaic viroid variant ls4b Ambros et al. 1998 J. Virol. 727397-7406 AJ005311 hammerhead hammerhead Peach latent mosaic viroidvariant ls16b Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005312hammerhead hammerhead Peach latent mosaic viroid variant ls17b Ambros etal. 1998 J. Virol. 72 7397-7406 AJ005313 hammerhead hammerhead Peachlatent mosaic viroid variant ls1 Ambros et al. 1998 J. Virol. 727397-7406 AJ005314 hammerhead hammerhead Peach latent mosaic viroidvariant ls18b Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005315hammerhead hammerhead Peach latent mosaic viroid variant ls11 Ambros etal. 1998 J. Virol. 72 7397-7406 AJ005316 hammerhead hammerhead Peachlatent mosaic viroid variant ls8 Ambros et al. 1998 J. Virol. 727397-7406 AJ005317 hammerhead hammerhead Peach latent mosaic viroidvariant ls19b Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005318hammerhead hammerhead Peach latent mosaic viroid variant ls5b Ambros etal. 1998 J. Virol. 72 7397-7406 AJ005319 hammerhead hammerhead Peachlatent mosaic viroid variant ls11b Ambros et al. 1998 J. Virol. 727397-7406 AJ005320 hammerhead hammerhead Peach latent mosaic viroidvariant ls6b Ambros et al. 1998 J. Virol. 72 7397-7406 AJ005321hammerhead hammerhead Peach latent mosaic viroid variant ls14b Ambros etal. 1998 J. Virol. 72 7397-7406 AJ005322 hammerhead hammerheadCrysanthemum chlorotic mottle viroid Navarro and 1997 Proc. Natl. Acad.Sci. 94 Y14700 hammerhead hammerhead Flores 11262-11267 Barley yellowdwarf virus satellite Miller et al. 1991 Virology 183 711-720 M63666hammerhead hammerhead RNA Arabis mosaic virus satellite Kaper et al.1988 Biochem. Biophys. Res. Com. M21212 hammerhead hairpin RNA 154318-325 Chicory yellow mottle Rubino et al. 1990 J. Gen Virol. 711897-1903 D00721 hammerhead hairpin virus satellite RNA S1 Lucernetransient streak Keese et al. 1983 FEBS Lett. 159 185-190 X01985hammerhead hammerhead virus satellite RNA LTSV-N Lucerne transientstreak Keese et al. 1983 FEBS Lett. 159 185-190 X01984 hammerheadhammerhead virus satellite RNA LTSV-A Lucerne transient streakAbouhaldar 1988 J. Gen. Virology 69 2369-2373 D00341 hammerheadhammerhead virus satellite RNA LTSV-C and Paliwal Tobacco ringspot virusBuzayan et al. 1986 Virology 151, 186-199 M14879 hammerhead hairpinsatellite RNA.1 Tobacco ringspot virus Buzayan et al. 1987 Virology 160,95-99 M17439 hammerhead hairpin satellite RNA.2 Subterraneanclovermottle Davies et al. 1990 Virology 177, 216-224 M33001 hammerhead virussatellite RNA.1 Subterraneanclover mottle Davies et al. 1990 Virology177, 216-224 M33000 hammerhead virus satellite RNA.2 Solanum nodiflorummottle virus Haseloff 1982 Nucleic Acids Res. 10 3681-3691 J02386hammerhead RNA and Symons Velvet tobacco mottle virus Haseloff 1982Nucleic Acids Res. 10 3681-3691 hammerhead circular viroid-like RNA-1and Symons Velvet tobacco mottle virus Haseloff 1982 Nucleic Acids Res.10 3681-3691 J02439 hammerhead circular viroid-like RNA-2 and SymonsCherry small circular viroid- Di Serio et al. 1997 J. Virol. 716603-6610 Y12833 mod. mod. like RNA hammerhead Carnation smallviroid-like RNA-1 Hernandez et al. 1992 Nucleic Acids Res. 20 6323-6329X68034 hammerhead hammerhead Carnation small viroid-like RNA-2 Hernandezet al. 1992 Nucleic Acids Res. 20 6323-6329 hammerhead hammerheadNotophtalmus viridescens Epstein et al. 1986 J. Cell. Biol. 1031137-1144 X04478 hammerhead (Newt) satellite 2 transcript Neurospora VSRNA Saville and 1990 Cell 61 685-696 M32974 VS RNA Collins selfcleavageSchistosome satellite DNA Ferbeyre et al. 1998 Mol. Cell. Biol. 183880-3888 AF036739

The use of ribozymes in transgenic organisms to generate RNA moleculeswith 5′ and or 3′ termini of interest has been documented in the art.Rubio et al. 1999, describe broad-spectrum protection againstTombusviruses elicited by defective interfering (D1) RNAs in transgenicplants. To produce RNAs with authentic 5′ and 3′ termini identical tothose of native Dl RNA, the Dl RNA sequence transcribed from a DNAcassette was flanked by ribozymes. Transgenic Nicotiana benthamianaplants were better protected than non-transgenic plants againstinfection by tomato bushy stunt virus and related tombusviruses. Dl RNAsinterfere drastically with virus accumulation through effectivecompetition with the parental virus for transacting factors required forreplication. Egli and Braus, 1994 describe uncoupling of mRNA 3′cleavage and polyadenylation by expression of a hammerhead ribozyme inyeast. Eckner et al. 1991 described that test gene transcripts whichcould obtain a mature histone 3′ end by the RNA cleaving activity of acis-acting ribozyme, thus circumventing the cellular 3′ end processingmachinery were found to be transport deficient and accumulated in thenuclear compartment. However, these documents in the art are not relatedto methods for inhibiting phenotypic expression by homology dependentgene-silencing, particularly by PTGS.

A particularly preferred self-splicing ribozyme is the ribozymecomprised with the Barley yellow dwarf virus (BYDV) satellite RNA, quiteparticularly the satellite RNA found in BYDV isolates of the RPVserotype.

It has been found that reduction of the phenotypic expression of thenucleic acid of interest using a chimeric gene according to theinvention was most efficient using a cDNA copy of the ribozyme comprisedwithin the minus strand of BYDV satellite RNA. Therefore, ribozymeswhich show an autocatalytic activity similar to the autocatalyticactivity of the ribozyme comprised within the minus strand of BYDVsatellite RNA are especially suited for the methods of the invention.Autocatalytic activity of ribozymes can be compared with theautocatalytic activity of the (−) strand of BYDV satellite RNA asdescribed by Miller et al. 1991.

The ribozyme motif within the (−) strand of BYDV satellite RNA has beenidentified as the nucleotide sequence of SEQ ID No 1 from the nucleotideat position 194 to the nucleotide at position 236. The ribozyme motifwithin the (+) strand of BYDV satellite RNA has been identified as thenucleotide sequence of SEQ ID No 2 from the nucleotide at position 310to the nucleotide at position 322 followed by the nucleotide sequence ofSEQ ID No. 2 from the nucleotide at position 1 to the nucleotide atposition 89.

It goes without saying that more than one DNA region encoding a ribozymemay be comprised within the chimeric gene. These ribozymes may beclustered, e.g. they may all be located the region immediatelyproceeding DNA region encoding the ′3 end formation and polyadenylationsignal.

However, it is expected that more than one DNA region encoding aribozyme may be comprised within the chimeric gene in such a way thatupon self-cleavage more than one unpolyadenylated RNA molecules eachcomprising a target-specific nucleotide sequence is generated. Such achimeric DNA could thus comprise:

a plant expressible promoter

a first target-specific DNA region

a DNA region encoding a first self-splicing ribozyme

a second target-specific DNA region

a DNA region encoding a second self-splicing ribozyme

a DNA region encoding a 3′ end formation and polyadenylation signal.

The first and second self-splicing ribozyme may be identical,essentially similar or different. Likewise, the first and secondtarget-specific DNA region encoding the RNA with a target-specificnucleotide sequence may be identical, essentially similar or different.

For practical reasons, it is thought that the number of DNA regionsencoding a ribozyme within a single chimeric gene should not exceedfive.

In a preferred embodiment, the nucleic acid of interest, whosephenotypic expression is targeted to be reduced, is a gene incorporatedin the genome of a eukaryotic cell, particularly a plant cell. It willbe appreciated that the means and methods of the invention can be usedfor the reduction of phenotypic expression of a gene which belongs tothe genome of the cell as naturally occurring, (an endogenous gene), aswell as for the reduction of phenotypic expression of a gene which doesnot belong to the genome of the cell as naturally occurring, but hasbeen introduced in that cell (a transgene). The transgene can beintroduced stably or transiently, and can be integrated into the nucleargenome of the cell, or be present on a replicating vector, such as aviral vector.

In another preferred embodiment, the nucleic acid of interest, whosephenotypic expression is targeted to be reduced is a viral nucleic acid,particularly a viral RNA molecule, capable of infecting a eukaryoticcell, particularly a plant cell. In this case, the phenotype to bereduced is the replication of the virus, and ultimately, the diseasesymptoms caused by the infecting virus.

For the purpose of the invention, the term “plant-expressible promoter”means a promoter which is capable of driving transcription in a plantcell. This includes any promoter of plant origin, but also any promoterof non-plant origin which is capable of directing transcription in aplant cell. A whole range of plant expressible promoters, is availableto direct the transcription of the chimeric genes of the invention.These include, but, are not limited to strong promoters such as CaMV35Spromoters (e.g., Harpster at al., 1988). In the light of the existenceof variant forms of the CaMV35S promoter, as known by the skilledartisan, the object of the invention can equally be achieved byemploying these alternative CaMV35S promoters and variants. It is alsoclear that other plant-expressible promoters, particularly constitutivepromoters, such as the opine synthase promoters of the Agrobacterium Ti-or Ri-plasmids, particularly a nopaline synthase promoter, orsubterranean clover virus promoters can be used to obtain similareffects. Also contemplated by the invention are chimeric genes to reducethe phenotypic expression of a nucleic acid in a cell, which are underthe control of single subunit bacteriophage RNA polymerase specificpromoters, such as a T7 or a T3 specific promoter, provided that thehost cells also comprise the corresponding RNA polymerase in an activeform.

It is a further object of the invention, to provide methods for reducingthe phenotypic expression of a nucleic acid in specific cells,particularly specific plant cells by placing the chimeric genes of theinvention under control of tissue-specific or organ-specific promoters.Such tissue-specific or organ-specific promoters are well known in theart and include but are not limited to seed-specific promoters (e.g.,W089/03887), organ-primordia specific promoters (An et al., 1996),stem-specific promoters (Keller et al., 1988), leaf specific promoters(Hudspeth et al., 1989), mesophyl-specific promoters (such as thelight-inducible Rubisco promoters), root-specific promoters (Keller etal., 1989), tuber-specific promoters (Keil et al., 1989), vasculartissue specific promoters (Peleman et al., 1989), stamen-selectivepromoters (WO 89/10396, WO 92/13956), dehiscence zone specific promoters(WO 97/13865) and the like.

In another embodiment of the invention, the expression of a chimericgene to reduce the phenotypic expression of a target nucleic acid can becontrolled at will by the application of an appropriate chemicalinducer, by operably linking the transcribed DNA region of the chimericgenes of the invention to a promoter whose expression is induced by achemical compound, such as the promoter of the gene disclosed inEuropean Patent publication (“EP”) 0332104, or the promoter of the genedisclosed in WO 90/08826.

It will be clear to the person skilled in the art that the same effectin reducing the phenotypic expression of a nucleic acid in a plant cellmay be achieved using a trans-splicing ribozyme to remove at least thepolyadenylation site from the RNA transcript of a chimeric genecomprising a plant expressible promoter, a target-specific DNA regionand a DNA region encoding a 3′ end termination and polyadenylationsignal to generate unpolyadenylated RNA comprising a target-specificnucleotide sequence.

As used herein “a trans-splicing ribozyme” is an RNA molecule capable ofcatalyzing the breakage or formation of a covalent bond within anotherRNA molecule at a specific site. The trans-splicing ribozyme should bechosen or designed in such a way that it recognizes a specific sitepreceding, preferably immediately preceding the polyadenylation signalof the RNA transcript comprising a target-specific nucleotide sequence.Methods to design such trans-splicing ribozyme with endoribonucleaseactivity are known in the art (see e.g. Haselhoff and Gerlach, 1988, WO89/05852)

The DNA region encoding a trans-splicing ribozyme may be comprisedwithin the chimeric gene encoding the target-specific RNA. Upontranscription of the chimeric gene an RNA molecule comprising thetrans-splicing ribozyme and the target-specific nucleotide sequence maythen generated, wherein the trans-splicing ribozyme is capable ofcleaving a specific site preceding the polyadenylation site of anothersimilar RNA molecule, to generate unpolyadenylated target-specific RNAmolecules.

The trans-splicing ribozyme may also be provided by expression ofanother chimeric gene encoding an RNA molecule comprising thetrans-splicing ribozyme in the same plant cell, according to methods andmeans available in the art (see e.g. Vaish et al. 1998; Bramlage et al.1998).

Alternative methods may exist to provide unpolyadenylatedtarget-specific RNA to the nucleus of a plant cell. Such methods includee.g. transcription of a chimeric gene, integrated in the nuclear genomeof a plant cell comprising a target-specific DNA region, by anDNA-dependent RNA polymerase different from RNA polymerase II, such thatRNA transcripts are generated independent from the normal processingmRNA machinery (including intron-splicing, capping and polyadenylation).This can be achieved e.g. by operably linking the target-specific DNAregion to a promoter region, recognized by a single subunit RNApolymerase from a bacteriophage, such as but not limited to the T7polymerase, and a DNA region comprising a terminator for such apolymerase. In this case, the plant cell needs to be provided with achimeric gene encoding the corresponding RNA polymerase. Providingunpolyadenylated target-specific RNA to the nucleus of a plant cell canalso be achieved e.g. by operably linking the target-specific DNA regionto a promoter region, recognized by a eukaryotic RNA polymerase I orIII, and a DNA region comprising a terminator for such a polymerase. Themeans and methods for constructing such chimeric genes and plant cellsare described in detail in WO 97/49814 (incorporated by reference).Another alternative to provide unpolyadenylated target-specific RNA tothe nucleus of a plant cell may include transcription of a chimeric genecomprising a target-specific DNA region operably linked to aplant-expressible promoter and linked to a DNA region comprising a 3′end formation signal but not a polyadenylation signal.

Although not intending to limit the invention to a specific mode ofaction, it is expected that the trigger of the homology-dependentgene-silencing mechanisms of the cell, particularly the co-suppressionmechanism, is the accumulation of target-specific RNA into the nucleusof that cell. Providing unpolyadenylated RNA to the nucleus of the cellmay be one mechanism of causing accumulation of target-specific RNA in anucleus of a cell, but other aberrations such as the absence of acap-structure or the presence of persistent introns etc. may constitutealternative ways to cause the accumulation of target-specific RNA in thenucleus of a cell.

Moreover, it is expected that other aberrations in the target-specificRNA molecules in addition to the absence of the polyA tail, includingthe absence of a cap-structure, or the presence of persistent introns orthe presence of abnormal secondary structures, particularly the presenceof giant hairpin structures, may have a cumulative effect on theinhibition of the normal transit of the RNA from the nucleus to thecytoplasm and hence have a cumulative or synergystic effect on thereduction of the phenotypic expression of a nucleic acid of interest.

The recombinant DNA comprising the chimeric gene to reduce thephenotypic expression of a nucleic acid of interest in a host cell, maybe accompanied by a chimeric marker gene, particularly when the stableintegration of the transgene in the genome of the host cell isenvisioned. The chimeric marker gene can comprise a marker DNA that isoperably linked at its 5′ end to a promoter, functioning in the hostcell of interest, particularly a plant-expressible promoter, preferablya constitutive promoter, such as the CaMV35S promoter, or a lightinducible promoter such as the promoter of the gene encoding the smallsubunit of Rubisco; and operably linked at its 3′ end to suitable planttranscription 3′ end formation and polyadenylation signals. It isexpected that the choice of the marker DNA is not critical, and anysuitable marker DNA can be used. For example, a marker DNA can encode aprotein that provides a distinguishable colour to the transformed plantcell, such as the Al gene (Meyer et al., 1987), can provide herbicideresistance to the transformed plant cell, such as the bar gene, encodingresistance to phosphinothricin (EP 0,242,246), or can provide antibioticresistance to the transformed cells, such as the aac(6′) gene, encodingresistance to gentamycin (WO94/01560).

A recombinant DNA comprising a chimeric gene to reduce the phenotypicexpression of a gene of interest, can be stably incorporated in thenuclear genome of a cell of a plant. Gene transfer can be carried outwith a vector that is a disarmed Ti-plasmid, comprising a chimeric geneof the invention, and carried by Agrobacterium. This transformation canbe carried out using the procedures described, for example, in EP 0 116718.

Alternatively, any type of vector can be used to transform the plantcell, applying methods such as direct gene transfer (as described, forexample, in EP 0 233 247), pollen-mediated transformation (as described,for example, in EP 0 270 356, W085/01856 and U.S. Pat. No. 4,684,611),plant RNA virus-mediated transformation (as described, for example, inEP 0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediatedtransformation (as described, for example, in U.S. Pat. No. 4,536,475),and the like.

Other methods, such as microprojectile bombardment as described for cornby Fromm et al. (1990) and Gordon-Kamm et al. (1990), are suitable aswell.

Cells of monocotyledonous plants, such as the major cereals, can also betransformed using wounded and/or enzyme-degraded compact embryogenictissue capable of forming compact embryogenic callus, or wounded and/ordegraded immature embryos as described in W092/09696. The resultingtransformed plant cell can then be used to regenerate a transformedplant in a conventional manner.

The obtained transformed plant can be used in a conventional breedingscheme to produce more transformed plants with the same characteristicsor to introduce the chimeric gene for reduction of the phenotypicexpression of a nucleic acid of interest of the invention in othervarieties of the same or related plant species; or in hybrid plants.Seeds obtained from the transformed plants contain the chimeric genes ofthe invention as a stable genomic insert.

The means and methods of the invention can also be used for thereduction, of gene expression by co-suppression in eukaryotic cells andorganisms.

In one embodiment the invention provides a method for reducing thephenotypic expression of a nucleic acid of interest, which is normallycapable of being expressed in a eukaryotic cell, comprising the step ofproviding unpolyadenylated RNA comprising a target specific sensenucleotide sequence of at least 10 consecutive nucleotides with at leastabout 70% sequence identity to about 100% sequence identity to thenucleotide sequence of the nucleic acid of interest, to the nucleus ofthe eukaryotic cell.

In another embodiment, a method is provided for reducing the phenotypicexpression of a nucleic acid of interest, which is normally capable ofbeing expressed in a eukaryotic cell, comprising the step of introducinginto the nuclear genome of the eukaryotic cell a chimeric DNA togenerate a transgenic plant cell, DNA comprising the following operablylinked parts:

-   -   (e) a promoter region functional in the eukaryotic cell;    -   (f) a target-specific DNA region comprising nucleotide sequence        of at least 10 consecutive nucleotides with at least about 70%        sequence identity to about 100% sequence identity to the        nucleotide sequence of the nucleic acid of interest;    -   (g) a DNA region encoding a self-splicing ribozyme; and    -   (h) a DNA region involved in 3′ end formation and        polyadenylation wherein the chimeric DNA when transcribed        produces a first RNA molecule comprising a target specific        nucleotide sequence and a self-splicing ribozyme, which when        cleaved by autocatalysis produces a second RNA molecule        comprising a target specific nucleotide sequence wherein the 3′        end of the first RNA molecule comprising the polyadenylation        site has been removed.

Different preferred embodiments and definitions described in connectionwith the reduction of gene expression by homology dependent genesilencing in plant cells and plants also apply mutatis mutandis to themeans and methods described for reduction of gene expression byco-suppression in eukaryotic cells and organisms. As used herein“eukaryotic cells” comprise plant cells, animal cells and human cellsand cells from yeasts and fungi as well as cultures of such cells.

It is a further object of the invention to provide eukaryotic cells,preferably plant cells and organisms (preferably plants) comprising thechimeric genes for the reduction of the phenotypic expression of atarget nucleic acid as described in the invention.

The methods and means of the invention can thus be used to reducephenotypic expression of a nucleic acid in a eukaryotic cell ororganism, particularly a plant cell or plant, for obtaining shatterresistance (WO 97/13865), for obtaining modified flower colour patterns(EP 522 880, U.S. Pat. No. 5,231,020), for obtaining nematode resistantplants (WO 92/21757, WO 93/10251, WO 94/17194), for delaying fruitripening (WO 91/16440, WO 91/05865, WO 91/16426, WO 92/17596, WO93/07275, WO 92/04456, U.S. Pat. No. 5,545,366), for obtaining malesterility (WO 94/29465, WO 89/10396, WO 92/18625), for reducing thepresence of unwanted (secondary) metabolites in organisms, such asglucosinofates (WO 97/16559) or chlorophyll content (EP 779 364) inplants, for modifying the profile of metabolites synthesized in aeukaryotic cell or organisms by metabolic engineering e.g. by reducingthe expression of particular genes involved in carbohydrate metabolism(WO 92/11375, WO 92/11376, U.S. Pat. No. 5,365,016, WO 95/07355) orlipid biosynthesis (WO 94/18337, U.S. Pat. No. 5,530,192) for delayingsenescence (WO 95/07993), for altering lignification in plants (WO93/05159, WO 93/05160), for altering the fibre quality in cotton (U.S.Pat. No. 5,597,718), for increasing bruising resistance in potatoes byreducing polyphenoloxidase (WO 94/03607), etc. The methods of theinvention will lead to better results and/or higher efficiencies whencompared to the methods using conventional sense or antisense nucleotidesequences and it is believed that other sequence-specific mechanismsregulating the phenotypic expression of target nucleic acids might beinvolved and/or triggered by the presence of the double-stranded RNAmolecules described in this specification.

A particular application for reduction of the phenotypic expression of atransgene in a plant cell, inter alfa, by antisense or sense methods,has been described for the restoration of male fertility, the latterbeing obtained by introduction of a transgene comprising a malesterility DNA (WO 94/09143, WO 91/02069). The nucleic acid of interestis specifically the male sterility DNA. Again, the processes andproducts described in this invention can be applied to these methods inorder to arrive at a more efficient restoration of male fertility.

It will be appreciated that the methods and means described in thespecification can also be applied in High Throughput Screening (HTS)methods, for the identification or confirmation of phenotypes associatedwith the expression of a nucleic acid sequence with hithertounidentified function in a eukaryotic cell, particularly in a plantcell.

-   -   Such a method comprises the steps of:    -   1. selecting a target sequence within the nucleic acid sequence        of interest with unidentified or non-confirmed        function/phenotype when expressed. Preferably, if the nucleic        acid has putative open reading frames, the target sequence        should comprise at least part of one of these open reading        frames. The length of the target nucleotide sequence may vary        from about 10 nucleotides up to a length equaling the length (in        nucleotides) of the nucleic acid of interest with unidentified        function.    -   2. Introducing a chimeric DNA into the nucleus of a suitable        host cell, comprising the nucleic acid of interest, wherein the        chimeric DNA comprises a promoter region suitable for expression        in the host cell, a DNA region encoding the target-specific        nucleotide sequence, and a DNA region encoding a self-splicing        ribozyme located immediately upstream of a DNA region involved        in 3′ end formation and polyadenylation.    -   3. observing the phenotype by a suitable method. Depending on        the phenotype expected, it may be sufficient to observe or        measure the phenotype in a single cell, but it may also be        required to culture the cells to obtain an (organized)        multicellular level, or even to regenerate a transgenic        organism, particularly a transgenic plant.

It is also clear that the methods and means of the invention are suitedfor the reduction of the phenotypic expression of a nucleic acid in allplant cells of all plants, whether they are monocotyledonous ordicotyledonous plants, particularly crop plants such as but not limitedto corn, rice, wheat, barley, sugarcane, cotton, oilseed rape, soybean,vegetables (including chicory, brassica vegetables, lettuce; tomato),tobacco, potato, sugarbeet but also plants used in horticulture,floriculture or forestry. The means and methods of the invention will beparticularly suited for plants which have complex genomes, such aspolyploid plants.

It is expected that the chimeric RNA molecules produced by transcriptionof the chimeric genes described herein, can spread systemicallythroughout a plant, and thus it is possible to reduce the phenotypicexpression of a nucleic acid in cells of a non-transgenic scion of aplant grafted onto a transgenic stock comprising the chimeric genes ofthe invention (or vice versa) a method which may be important inhorticulture, viticulture or in fruit production.

The following non-limiting Examples describe the construction ofchimeric genes for the reduction of the phenotypic expression of anucleic acid of interest in a eukaryotic cell and the use of such genes.Unless stated otherwise in the Examples, all recombinant DNA techniquesare carried out according to standard protocols as described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 ofAusubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA. Standard materials and methods for plant molecular workare described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,jointly published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK.

Throughout the description and Examples, reference is made to thefollowing sequences:

SEQ ID No 1: cDNA copy of the (−) strand of BYDV RPV satellite, RNA

SEQ ID No 2: cDNA copy of the (+) strand of BYDV RPV satellite RNA

SEQ ID No 3: oligonucleotide for PCR amplification (SatPR1)

SEQ ID No 4: oligonucleotide for PCR amplification (SatPR2)

SEQ ID No 5: oligonucleotide for PCR amplification (SatPR3)

SEQ ID No 6: oligonucleotide for PCR amplification (SatPR4)

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should not be construed,however, as limiting the broad scope of the invention.

EXAMPLES Example 1 Experimental Procedures

1.1 Chimeric DNA Constructs

Ribozyme-Containing GUS Gene Constructs and a Control Construct

The ribozyme sequences used are the plus strand or negative strandself-cleavage sequences of the satellite RNA of the barley yellow dwarfvirus (BYDV) RPV serotype, which was isolated in CSlRO Plant Industry(SEQ ID 1 and 2; Miller et al., 1991).

The two ribozyme-containing GUS constructs (pMBW259 and pMBW267) and onecontrol GUS construct (pMBW265) are schematically drawn in FIG. 1.pMBW259 contains two plus strand cleavage sites, while pMBW267 containsthe negative strand cleavage site.

To make these constructs, a β-glucuronidase (GUS) gene sequence wasmodified to contain a NcoI site around the translational start ATG andcloned into pART7 (Gleave, 1992) at the Xhol/EcoRI sites, formingpMBW258. The full-length BYDV-RPV satellite sequence was amplified byPCR using primers SatPR1 (SEQ ID No. 3) and SatPR4 (SEQ ID No. 6),digested with BamHl and cloned into pMBW258 at the BamHl site, and theresulting 35S-GUS-Sat-ocs cassette was excised and cloned into pART27(Gleave, 1992), forming pMBW265. The same full-length satellite sequencewas inserted into the BamHl site of pMBW258 but in the antisenseorientation, and the resulting 35S-GUS-asSat-ocs was cloned into pART27to give rise to pMBW267.

To make pMBW259, the 3′ and 5′ halts of the satellite RNA sequences wereamplified by PCR using primer pairs SatPR3 (SEQ ID No. 5) and SatPR4(SEQ ID No. 6), and using SatPRl (SEQ ID No. 3).and SatPR2 (SEQ ID No4), respectively. Fusion of the full-length sequence with the 3′ halfand the 5′ half sequences were made through ligation between the EcoRVand HpaI ends of the three PCR fragments. This fusion mimics the naturalmultimeric forms of the satellite RNA, and therefore maintains the plusstrand cleavae property of the native forms. The fusion sequence wascloned into pGEM-3Z (Promega) at the SacI/PstI sites, excised withHindIII/EcoRI, blunted, and inserted into pART7 at the SmaI site, intowhich the GUS sequence described above was then cloned at the XhoI/EcoRIsites. The resulting 3SS-GUS-Sat-ocs was inserted into pART27 at theNotI site, forming pMBW259.

The Super-Transforming GUS Construct

The BamHI fragment was excised from pIG121 Hm (Hiei et al.,1994) andcloned into pART7. The GUS-nos sequence was then excised by Accl,blunted, and inserted into pBluescript at the HincII site. The 1.3 kbregion of a cucurbit phloem protein PP2 gene was excised withNotI/HindIII from a lambda clone CPPI.3 and cloned into the aboveBluescript plasmid. The resulting PP2-GUS-nos was excised with NotI/Kpnland inserted into pWBVec2 (Wang et al., 1998), giving rise to pBPPGH(FIG. 1).

1.2 Tobacco Transformation

Nicotiana tobaccum cv. W38 was transformed and regenerated into wholeplants essentially as described by Ellis et al. 1987. For constructspMBW259, pMBW265 and pMBW267, 50 mg/L kanamycin was included in themedia for selection of transformed tissue. For construct pBPPGH, 25 mg/Lhygromycin B was used.

1.3 GUS Assay

GUS gene expression was assayed histochemically or fluorometricallyaccording to Jefferson et al. 1987.

Example 2 GUS Expression in Transgenic Tobacco Transformed with a SingleType of the GUS Constructs.

Transgenic plants containing pMBW259 and pMBW267 showed very low levelsof GUS expression, as judged by lack of, or faint blue, GUS staining.Plants transformed with pMBW265 showed more GUS expression than withpMBW259 and pMBW267, but the level was much lower than plantstransformed with pBPPGH. The best pMBW265 lines expressed 13.3% of theGUS activity by an average pBPPGH line.

Example 3 GUS Expression in Super-Transformed Lines Containing pBPPGHand One of the Three Other Constructs of Example 1

In order to promote silencing of a normal GUS gene by the presence ofthe ribozyme sequence near the 3′ end of the GUS gene transcript, plantscontaining pMBW259, pMBW265 or pMBW267 and pBPPGH were constructed byre-transformation. Histochemical GUS assays of the super-transformantsshowed that the pMBW267 background gave substantially higher proportionsof transformants than the pMBW259 or the pMBW265 background that showedlow levels of GUS expression as indicated by the lack of strong anduniform blue staining. Super-transformants containing pBPPGH and pMBW265showed the best GUS expression.

Table 2 shows the result of fluorometric GUS (MUG) assay of thesupertransformants. The lines (E and F) containing pBPPGH and pMBW267showed uniformly low GUS expression compared with the other lines. Thebest GUS expression came from the C lines which contain pBPPGH andpMBW265.

Among the three constructs tested, pMBW265 does not contain thefull-length functional ribozyme sequences of the BYDV satellite RNA in acontinuous stretch, and is therefore expected to produce mainlypoly(A)+RNA. pMBW259 contains two copies of the plus strand ribozymesequence, and should give rise to RNA that have poly(A) tails removed byribozyme cleavage. pMBW267 contain the negative strand ribozyme. Thenegative strand ribozyme was previously shown to be much (at least10-fold) more efficient than the plus strand ribozyme (Miller et al.,1991), and therefore it is expected that pMBW267 produces poly(A)-RNAmore efficiently. Our experiment showed that the super-transformed lineshaving the pMBW267 background expressed uniformly low levels of GUSactivity in comparison with the lines having the pMBW259 or the pMBW265background. The highest GUS expressing lines were from the pMBW265background, which does not produce polyA-RNA.

TABLE 2 MUG assay of super-transformed tobacco lines*. Super-transformed MUG lines Readings A1 10.1 A2 15.8 A3 30.6 A4 47.3 A5 0.29A6 10.3 A7 5.8 A8 13.15 A9 7.34 A10 9.76 A11 17.74 A12 34.8 A13 4.33 A143.41 A15 11.2 A16 2.04 A17 13.29 A18 14.6 A19 0.14 A20 17.2 A21 9.22 A2217.3 B1 9.57 B2 44.7 B3 17.7 B4 1.25 B5 13.5 B6 11.4 B7 6.28 B8 24.8 B916.3 B10 9.72 B11 3.71 B12 0.08 B13 20.6 B14 11.9 B15 3.11 B16 8.25 B174.12 B18 6.04 C1 8.84 C2 16.9 C3 17.9 C4 22.8 C5 11.7 C6 14.5 C7 44.0 C819.0 C9 29.8 C10 32.1 C11 37.1 C12 2.51 C13 14.5 C14 25.8 C15 7.20 C1630.2 C17 9.70 C18 13.4 C19 19.3 C20 17.0 D1 6.01 D2 12.9 D3 0.19 D4 7.88D5 1.24 D6 0.44 D7 14.1 D8 0.91 D9 5.49 D10 1.30 D11 15.1 D12 6.63 D1312.2 D14 15.8 D15 1.32 D16 2.29 D17 3.59 D18 22.1 D19 13.0 D20 4.37 E14.32 E2 3.15 E3 3.56 E4 3.31 E5 3.68 E6 5.02 E7 2.63 E8 10.27 E9 10.81E10 13.1 E11 5.10 E12 2.86 E13 4.00 E14 16.8 E15 4.02 E16 1.29 E17 1.78E18 3.57 E19 0.43 E20 11.8 F1 5.73 F2 5.10 F3 4.16 F4 4.69 F5 0 F6 1.93F7 3.21 F8 2.77 F9 1.86 F10 3.27 F11 2.85 F12 3.25 F13 2.17 F14 2.84 F153.11 F16 2.06 F17 2.90 F18 3.75 F19 4.16 F20 2.49 *A and B, fromsuper-transformation of two independent pMBW259 lines with pBPPGH; C andD, from super-transformation of two independent pMBW265 lines withpBPPGH; E and F, from super-transformation of two independent pMBW267lines with pBPPGH.

Example 4

Experimental Procedures

Gene Construction

Standard gene cloning methods (Sambrook et al. 1989) were used to makethe chimeric genes. A schematic representation of the constructs used isshown in FIGS. 2A and 2B.

The components for these constructs were:

Cauliflower mosaic virus 35S promoter from the Cabb-JI isolate(35S)(Harpster et al., 1988)

Octopine synthase terminator (ocs-t) (MacDonald et al., 1991)

Subterranean clover virus promoter No 4 (S4) (WO 9606932)

Subterranean clover virus terminator No 4 (s4t) (WO 9606932)

Subterranean clover virus double enhancer promoter No 4 (S4S4)

Subterranean clover virus promoter No 4 with S7 enhancer (S7S4)

Maize ubiquitin promoter (Ubi) (Christensen and Quail, 1996)

Agrobacterium tumour morphology 1 gene terminator (tm1′) (Zheng et al.,1991)

the Nia gene of an Australian strain of Potato virus Y (Nia)

a dysfunctional β-glucuronidase open reading frame encoding DNA (Gusd)

a modified 5′ untranslated region (5′UTR) from Johnsongrass mosaic virus(JGMV5′)

This contains insertion of a NcoI site at the ATG start codon followedby three stop codons in frame, and a PstI site (for insertion of theintron as in constructs 4 and 5 of FIG. 2A). In vector constructs 2 and6 of FIG. 2A, the Gusd open reading frame is inserted in at the NcoIsite, removing the stop codons; in all other constructs of FIG. 2A it isinserted downstream of the PstI site.

a castor bean catalase intron (Ohta et al., 1990) as modified by Wang etal. (1997) (“intron”).

The chimeric genes were constructed by operably assembling the differentparts as schematically indicated in FIG. 2A or FIG. 2B and inserting theobtained chimeric genes in the T-DNA vectors pART27 and pART7 vectors(Gleave, 1992) between the left T-DNA border and the chimeric plantexpressible neo gene.

The DNA encoding a dysfunctional β-glucuronidase open reading frame(GUSd) was obtained by deleting from a gus coding region the sequencebetween the two EcoRV restriction sites. For the construction of thechimeric gene encoding the RNA molecule comprising both sense andantisense nucleotide sequence to a β-glucuronidase gene, a sequence wasadded to the Gusd gene to be allow base pairing the 5′end over 558bases. This sequence was cloned between the maize ubiquitin promoter andthe tm1′ terminator and inserted in a T-DNA vector.

T-DNA vectors were constructed which comprised a first and a secondchimeric virus resistance gene, wherein the first chimeric geneconsisted of:

-   -   1. a CaMV 35S promoter sequence, coupled to    -   2. in sense orientation, the nucleotide sequence from PVY        encoding either        -   Vpg protein (see e.g., Genbank Accession Nr ZZ9526 from            nucleotide 1013 to nucleotide 1583), or        -   part of the CI protein (see e.g., Genbank Accession Nr            M95491 from nucleotide 3688 to nucleotide 4215) or        -   Protease (Pro) (see e.g., EMBL Accession Nr D00441 from            nucleotide 5714 to nucleotide 7009), followed by    -   3. the S4 terminator from subterranean clover mosaic virus, as        described above.

The second chimeric gene consists of

-   -   1. a S4 promoter as described above, coupled to    -   2. in anti-sense orientation, the nucleotide sequence from PVY        encoding either        -   Vpg protein, or        -   CI protein or        -   Protease, followed by    -   3. the octopine synthase terminator as described above.

The sense and antisense sequences within one T-DNA vector were derivedfrom the same PVY coding region.

Also, T-DNA vectors were constructed for use in altering the fatty acidcomposition in oil (see FIG. 3), comprising

-   -   1. a FPI promoter (truncated seed specific napin promoter,        containing sequences between −309 and +1, as described in        Stalberg et al; linked to    -   2. a nucleotide sequence comprising the 480 bp located 5′ in the        ORF encoding the Δ12 desaturase from Arabidopsis thaliana (Fad2)        in sense orientation and in antisense orientation, linked by a        623 bp spacer sequence; followed by    -   3. the terminator from the nopaline synthase gene.

In addition, T-DNA vectors were constructed to evaluate the influence ofa presence of an intron sequence in the chimeric genes encodingcomplementary pair (“CoP”) constructs. To this end, constructs were madecomprising:

-   -   1. a CamV35S promoter, followed by    -   2. the protease encoding ORF from PVY (see above) in sense        orientation;    -   3. the sequence of the Flaveria trinervia pyruvate        orthophosphate dikinase intron 2)    -   4. the protease encoding ORF from PVY in antisense orientation;        and    -   5. the octopine synthase gene terminator.

Plant Transformation

Nicotiana tobaccum (W38) tissue was transformed and regenerated intowhole plants essentially as described by Landsman et al. (1988). Rice(Oryza sativa) was transformed essentially as described by Wang et al.(1997).

Rice Supertransformation

Mature embryos from a rice plant expressing GUS and hygromycinphosphotransferase (HPT) activity were excised from mature seed andplaced on callus inducing media for 7 weeks. Calli were recovered fromthese cultures, incubated with Agrobacteria containing various binaryvector constructs for 2 days, then placed on callusing media containinghygromycin, bialaphos and Timentin™. During the next four weekshygromycin and bialaphos resistant calli developed. These callus lineswere maintained on hygromycin and bialaphos containing media for afurther 2 months before being assayed for GUS activity.

GUS Assay

Rice calli were tested for GUS activity using the histochemical stainX-glucuronide or the fluorogenic substrate 4-methyl-umbeliferoneglucuronide (MUG) essentially as described by Jefferson at al. (1987).

Comparison of Chimeric Genes Comprising Only Antisense, Only Sense, orBoth Sense and Antisense (Complimentary Pair (CoP)) Sequence forReduction in Phenotypic Expression of an Integrated β-glucuronidaseGene.

Transgenic rice tissue expressing β-glucuronidase (GUS) from a singletransgene (and hygromycin resistance from a hph gene) (lines V10-28 andV10-67) was supertransformed using vectors that contained the bar geneconferring phosphinothricin resistance and various sense, antisense andCoP constructs (see FIG. 2A) derived from a crippled GUS (GUSd) gene.The supertransformed tissue was maintained on hygromycin and bialaphosselection media for 3 weeks then analyzed for GUS activity. A crippledGUS gene was used so that expression from this gene would not besuperimposed on the endogenous GUS activity.

The figures in Table 2 represent the rate of MU production measured byabsorption at 455 nm, with excitation at 365 nm of 1.5 μg of totalprotein in a reaction volume of 200 μl. The rate was measured over 30min at 37° C. The reading for non-transgenic rice calli was 0.162. Thefigures in bracket which follow the description of the introducedconstruct refer to FIG. 2A.

The results (Table 2) showed that supertransformation with the binaryvector containing the bar gene without the GUSd gene had no silencingeffect on the endogenous GUS activity. Supertransformation with GUSd ina sense or antisense orientation, with or without an intron or an earlystop codon, showed some degree of reduction (in about 25% of theanalyzed calli) of the endogenous GUS activity (see last two rows inTable 2 representing the percentage of analyzed calli with a MUG assayreading of less than 2.000). However, supertransformation with a CoPconstruct gave in about 75% to 100% of the analyzed calli, reduction ofthe endogenous GUS activity. This CoP construct was designed so that the3′ end of the mRNA produced could form a duplex with the 5′ end of thetranscript to give a “panhandle” structure.

These data show that a complimentary pair can be made using oneself-annealing transcript, that this design is much more effective thana conventional sense or antisense construct, and that the approach canbe used to reduce the phenotypic expression of genes present in a plantcell.

TABLE 2 MUG assay of Supertransformed Rice Calli Sense + Antisense +Inverted Vector Sense + stop + stop + repeat cassette Sense Stop intronintron CoP (1) (2) (3) (4) (5) (6) V10-28 121.0 97.45 38.43 38.88 0.2900.565 45.58 6.637 64.16 115.5 0.572 0.316 99.28 71.60 149.2 133.0 37.20.351 26.17 0.224 0.955 98.46 53.94 0.210 92.21 0.321 68.32 0.502 105.50.701 108.8 5.290 105.6 39.35 56.73 0.733 6.432 0.9460 136.6 1.545 60.362.103 90.80 32.44 140.4 10.36 71.12 119.8 98.24 128.8 62.38 111.6 13.170.717 93.76 31.28 17.79 14.42 0.424 0.398 5.023 88.06 26.98 0.315 40.2752.28 115.5 0.270 36.40 30.26 149.7 16.78 53.24 107.5 66.75 67.28 29.9726.75 145.8 0.217 89.06 105.1 0.534 0.208 0.256 135.1 9.4 68.23 95.0435.33 5.481 71.5 V10-67 318.8 93.43 0.199 31.82 1.395 0.472 109.5 73.190.197 58.08 152.4 0.256 30.35 128.1 0.157 56.32 67.42 0.296 40.04 1.506128 44.62 12.11 0.452 228 140.6 130.3 0.454 0.668 0.422 23.05 1.275196.2 17.32 23.34 0.196 241.2 0.272 12.43 73.2 76.10 0.294 118.5 0.209140.0 20.32 130.1 0.172 11.27 42.05 90.13 107.4 0.841 0.436 110.6 117.5157.4 0.453 66.12 0.398 19.29 118.9 0.518 87.81 136.9 0.242 121.0 21.440.231 0.299 67.92 115.1 155.0 116.1 0.206 50.32 77.1 190.9 43.18 12.47170.3 106.1 0.773 31.06 0.213 108.9 73.12 0.146 11.15 1.241 29.97 19.224.092 50.11 169.6 80.34 76.88 117.8 22.08 159.1 91.6 67.52 7.855 92.3269.76 27.97 0.822 V10-28 0%   21% 10% 10.5% 22%  75% V10-67 0% 37.5% 33%29.5% 21% 100%

Example 5 Intron Enhanced Silencing

The T-DNA vectors comprising the chimeric genes encoding the CoPconstructs wherein an intron (Flaveria trinervia pyruvate orthophosphatedikinase intron 2) has been inserted in either the sense orientation orthe antisense orientation, between the sense and antisense sequencescorresponding to the protease encoding ORF from PVY (as described aboveand in PCT-application PCT/IB99/00606) were used to obtain transformedtobacco plants, which were subsequently challenged with PVY. The resultsare summarized in the following table:

TABLE 3 Number of immune plants/Number of Construct independenttransgenic plants 35S-Pro(sense)-intron(sense)- 22/24Pro(antisense)-Ocs-t 35S-Pro(sense)-intron(antisense)- 21/24Pro(antisense)Ocs-t

Example 6 Modifying Oil Profile Using CoP Constructs in Arabidopsis

T-DNA vectors for modifying the fatty acid composition in oil, extractedfrom crushed seeds as described above and in PCT-applicationPCT/IB99/00606 were used to introduce the chimeric gene encoding the CoPconstruct for reducing the expression (see FIG. 3A) the Δ12 desaturasegene (Fad2) in Arabidopsis thaliana.

For comparison of the efficiency, transgenic Arabidopsis plants weregenerated wherein the Fad2 gene expression was reduced by a plaincosuppression construct, comprising the FPI seed-specific promotercoupled to the complete ORF from the L12 desaturase gene (Fad2) inArabidopsis thaliana and the nopaline synthase promoter (see FIG. 3B).

As control plants, transgenic Arabidopsis transformed by unrelated T-DNAconstructs were used.

Seeds were harvested, crushed and extracted and the percentage of themajor fatty acids in the oil was determined by methods available in theart. The results, which are the mean of two readings, are summarized inTable 4.

TABLE 4 Peak Names Sample Myris- Palmi- Palmi- C18:1/ Name tic tictoleic Stearic Oleic Linoleic Linolenic 20:0 20:1 22:0 22:1 24:0(C18:2 + C18:3) Hairpin 1.1 0.00 6.06 0.52 3.21 56.65 7.50 6.82 1.4616.02 0.00 1.76 0.00 3.95 Hairpin 1.2 0.12 6.86 0.39 3.40 51.28 10.008.73 1.64 15.60 0.00 1.97 0.00 2.74 Hairpin 1.3 0.11 8.47 0.50 3.4921.64 28.99 18.51 2.02 14.19 0.00 2.09 0.00 0.46 Hairpin 1.4 0.00 6.140.50 3.37 51.70 9.77 8.02 1.73 16.04 0.00 2.05 0.67 2.91 Hairpin 2.10.06 5.19 0.43 3.33 54.84 5.52 7.76 1.77 18.50 0.34 1.83 0.45 4.13Hairpin 2.2 0.04 7.67 0.46 3.75 19.60 28.29 18.64 2.55 15.96 0.19 2.280.56 0.42 Hairpin 3.1 0.00 7.99 0.53 3.62 19.52 28.41 19.24 2.32 15.140.00 2.23 0.99 0.41 Hairpin 3.2 0.09 7.00 0.54 3.69 49.02 11.03 9.641.71 14.94 0.00 1.72 0.62 2.37 Hairpin 3.3 0.00 5.68 0.49 3.98 46.1912.82 9.71 2.10 16.70 0.00 1.94 0.39 2.05 Hairpin 3.4 0.17 7.19 0.773.69 45.90 11.86 10.65 1.84 15.39 0.00 1.90 0.65 2.04 Hairpin 3.5 0.006.45 0.48 3.26 51.76 8.13 10.04 1.51 16.08 0.00 1.92 0.36 2.85 Hairpin3.6 0.08 7.51 0.23 3.59 19.97 29.13 20.12 2.15 14.54 0.29 2.02 0.36 0.41Hairpin 3.7 0.14 7.20 0.78 2.90 26.37 24.81 17.18 1.92 15.50 0.36 2.300.53 0.63 Hairpin 3.8 0.11 6.34 0.46 3.23 38.58 15.25 13.54 1.89 16.910.00 2.36 1.34 1.34 Hairpin 3.9 0.00 6.47 0.49 3.32 47.59 11.44 9.631.68 15.96 0.00 1.88 1.55 2.26 Hairpin 3.10 0.00 6.77 0.56 3.48 53.307.57 9.34 1.55 15.65 0.00 1.79 0.00 3.15 Hairpin 3.11 0.00 7.05 0.593.61 53.62 8.87 8.36 1.55 14.35 0.00 1.99 0.00 3.11 Hairpin 3.12 0.058.32 0.36 3.85 18.48 29.24 19.94 2.48 14.75 0.00 2.28 0.26 0.38 Hairpin4.1 0.09 6.97 0.59 3.61 53.64 8.40 8.44 1.60 15.00 0.00 1.66 0.00 3.19Hairpin 4.2 0.07 6.81 0.22 3.27 55.06 9.16 8.71 1.26 13.63 0.19 1.330.30 3.08 Hairpin 4.3 0.04 6.81 0.50 3.47 46.21 10.67 11.52 1.81 16.500.00 1.88 0.58 2.08 Hairpin 5.1 0.00 8.30 0.23 3.71 17.72 28.92 20.632.38 14.77 0.00 2.41 0.92 0.36 Hairpin 5.2 0.19 7.15 1.55 3.56 44.5811.44 11.59 1.77 15.67 0.00 1.84 0.65 1.94 Hairpin 5.3 0.10 6.49 0.403.72 54.19 7.01 7.89 1.74 15.91 0.00 1.92 0.62 3.64 Hairpin 5.5 0.126.58 0.51 3.84 54.48 6.16 7.23 1.77 16.50 0.42 1.90 0.48 4.07 Hairpin5.6 0.00 6.67 0.50 3.66 46.32 11.56 10.48 1.83 15.99 0.00 2.15 0.84 2.10Hairpin 5.7 0.00 5.50 0.51 3.58 57.33 4.75 5.91 1.75 18.03 0.00 1.880.76 5.38 Hairpin 5.8 0.16 6.55 1.53 3.54 48.52 9.91 8.97 1.78 16.390.00 1.84 0.81 2.57 Hairpin 6.1 0.10 6.35 0.57 3.48 59.00 4.77 6.26 1.4815.95 0.00 1.80 0.25 5.35 Hairpin 6.2 0.10 7.98 0.37 4.06 20.96 29.0118.69 2.38 13.63 0.20 2.03 0.60 0.44 Hairpin 6.5 0.08 6.21 0.63 3.6160.05 5.07 5.27 1.55 15.20 0.00 1.69 0.66 5.81 Columbia pBin 19 0.088.81 0.47 3.51 17.07 30.31 20.94 1.78 14.56 0.00 2.17 0.28 0.33 controlCosuppresion 1.1 0.08 8.16 0.62 3.71 26.16 23.77 18.15 2.06 14.65 0.171.89 0.57 0.62 Cosuppresion 1.2 0.00 8.49 0.53 3.65 17.90 29.93 20.362.34 14.25 0.00 2.33 0.23 0.36 Cosuppresion 1.3 0.07 6.65 0.40 3.4238.34 15.25 14.16 1.91 17.19 0.31 1.94 0.35 1.30 Cosuppresion 1.4 0.008.22 0.57 3.82 18.27 28.82 19.63 2.56 14.83 0.00 2.46 0.83 0.38Cosuppresion 1.5 0.00 7.51 0.52 3.84 34.59 17.90 14.64 2.18 16.27 0.002.02 0.54 1.06 Cosuppresion 1.6 0.07 7.44 0.47 3.16 23.97 27.32 17.292.03 15.52 0.18 2.22 0.33 0.54 Cosuppresion 2.1 0.07 7.46 0.43 3.0023.91 27.21 17.79 1.84 15.27 0.30 2.14 0.58 0.53 Cosuppresion 2.2 0.008.19 0.55 4.22 18.59 28.31 18.80 2.77 15.51 0.00 2.46 0.58 0.39Cosuppresion 2.3 0.00 8.71 0.47 3.48 19.21 30.06 19.49 2.03 13.78 0.002.15 0.63 0.39 Cosuppresion 3.1 0.06 7.57 0.50 3.83 32.24 20.00 15.662.06 15.65 0.34 1.85 0.23 0.90 Cosuppresion 4.1 0.00 7.29 0.43 3.5530.26 21.17 17.06 2.01 16.08 0.00 1.92 0.25 0.79 Cosuppresion 4.2 0.088.02 0.53 3.62 33.04 20.04 15.68 1.80 14.72 0.00 1.88 0.58 0.92Cosuppresion 4.3 0.07 8.35 0.54 3.85 30.02 21.72 16.78 2.01 14.25 0.001.92 0.49 0.78 Cosuppresion 4.4 0.06 6.98 0.53 3.62 43.38 13.24 12.771.74 15.37 0.30 1.67 0.33 1.67 Cosuppresion 4.5 0.13 7.84 0.52 3.7633.76 18.16 16.21 1.89 14.96 0.35 1.85 0.57 0.98 Cosuppresion 4.6 0.118.18 0.32 3.58 19.72 29.19 20.26 2.04 13.92 0.29 1.84 0.55 0.40Cosuppresion 4.7 0.11 7.88 0.39 3.75 27.40 22.85 17.44 2.08 15.29 0.002.04 0.76 0.68 Cosuppresion 4.8 0.13 7.56 0.41 3.46 32.27 20.50 15.451.90 15.47 0.00 2.02 0.83 0.90 Cosuppresion 4.9 0.09 7.46 0.29 3.7536.11 16.96 15.74 1.92 15.38 0.31 1.74 0.25 1.10 Cosuppresion 5.1 0.107.68 0.34 3.88 36.00 16.77 15.38 1.90 15.44 0.32 1.82 0.36 1.12Cosuppresion 5.2 0.08 7.56 0.25 3.58 26.10 25.11 17.79 1.96 15.03 0.301.72 0.54 0.61 Cosuppresion 5.3 0.08 7.38 0.20 3.56 42.24 13.33 13.321.76 15.19 0.16 1.61 1.18 1.59 Cosuppresion 6.1 0.08 8.04 0.50 3.6831.37 20.29 17.17 1.84 14.31 0.00 1.76 0.95 0.84 Cosuppresion 6.2 0.008.50 0.51 3.91 18.59 29.33 19.66 2.46 14.75 0.00 2.28 0.00 0.38 Controlc24 pGNAP- 0.07 8.30 0.10 4.78 19.68 25.91 20.56 2.97 15.29 0.31 1.790.24 0.42 p450

Analysis of the results indicates that transgenic plants harboring a CoPconstruct (indicated as “hairpin x.x” in the table) have a higherfrequency of plants with oil wherein the increase in oleic acid andconcomitant decrease in linolenic and linoleic acid is significant thanin transgenic plants harboring cosuppression constructs. Moreover theabsolute levels of increase, respectively decrease are higherrespectively lower than in transgenic plants harboring cosuppressionconstructs.

Example 7 Modifying Oil Profile Using CoP Constructs in Brassica

The T-DNA vector harboring the chimeric gene encoding the CoP constructdescribed in Example 6 is introduced in Brassica oilseed rape. Seedsharvested from the transgenic Brassica sp. are crashed and oil extractedand the composition of the fatty acids in the oil is analyzed.

Oil from transgenic Brassica sp. harboring the CoP construct havesignificantly increased oleic acid content and decreased linoleic andlinolenic acid content. A T-DNA vector harboring a chimeric geneencoding a CoP construct similar to the one described in Example 6, butwherein the sequence of the sense and antisense region corresponding tothe Δ12 desaturase encoding ORF is based on a homologous ORF fromBrassica spp. is constructed and introduced in Brassica 20 oilseed rape.

The sequence of Brassica spp ORFs homologous to Δ12 desaturase encodingORF from Arabidopsis are available from Genbank database under Accessionnrs AF042841 and AF124360.

Seeds harvested from the transgenic Brassica sp. are crashed and oilextracted and the composition of the fatty acids in the oil is analyzed.Oil from transgenic Brassica sp. harbouring the CoP construct havesignificantly increased oleic acid content and decreased linoleic andlinolenic acid content.

Example 8 Suppression of an Endogenous Rust Resistance Gene in Flax

A CoP construct for suppression of the endogenous rust resistance genewas made consisting of

-   -   1. a CaMV35S promoter; operably linked to    -   2. part of an endogenous rust resistance gene (n) from flax        (about 1500 bp long) in the sense orientation; ligated to    -   3. a similar part of the endogenous rust resistance gene from        flax (about 1450 bp long) in antisense orientation so that a        perfect inverted repeat without spacer sequence is generated        wherein each repeat is about 1450 bp long; followed by    -   4. a nos terminator.

Plain antisense constructs were made using a similar fragment asdescribed sub 3 above inserted between a CaMV35S promoter and a nosterminator.

Flax plants containing the n gene (which gives resistance to a strain offlax rust) were transformed by these CoP and antisense constructs. Ifsuppression occurs, the plants become susceptible to the rust strain. Ifthe construct has no effect, the transformed plants remain resistant tothe rust strain.

Results

20 ngc-b sense/antisense 3 suppressed out of 7 ngc-b antisense 0suppressed out of 12

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1. (canceled)
 2. A chimeric DNA comprising a promoter operably linked toa target specific DNA region which encodes an unpolyadenylated hairpinRNA, wherein the target specific DNA region comprises a target specificsense nucleotide sequence and a target specific antisense nucleotidesequence, wherein the target specific antisense nucleotide sequencecomprises 20 consecutive nucleotides in a sequence identical to thesequence of a complement of a part of an RNA molecule transcribed orproduced from a nucleic acid of interest in an animal cell, wherein thetarget specific sense nucleotide sequence comprises 20 consecutivenucleotides in a sequence identical to the sequence of the part of theRNA molecule transcribed or produced from the nucleic acid of interest,wherein the target specific sense nucleotide sequence and the targetspecific antisense nucleotide sequence are separated and linked by aspacer sequence, such that the unpolyadenylated hairpin RNA resultingfrom transcription of the target specific DNA region comprises thespacer sequence located between sense and antisense nucleotide sequencesin the unpolyadenylated hairpin RNA, and the sense and antisensenucleotide sequences are complementary to each other so as to form theunpolyadenylated hairpin RNA.
 3. The chimeric DNA of claim 2, whereinsaid target specific sense nucleotide sequence corresponds to atranslated region of the nucleic acid of interest.
 4. The chimeric DNAof claim 2, wherein the target specific sense nucleotide sequencecorresponds to an untranslated region of the RNA molecule produced fromthe nucleic acid of interest.
 5. The chimeric DNA of claim 2, whereinthe promoter is recognized by a eukaryotic RNA polymerase I or III andthe DNA further comprises a terminator for the polymerase I or III. 6.The chimeric DNA of claim 2, wherein the nucleic acid of interest is agene incorporated in the genome of the animal cell.
 7. The chimeric DNAof claim 2, wherein the nucleic acid of interest is an endogenous geneof the animal cell.
 8. The chimeric DNA of claim 2, wherein the nucleicacid of interest is a viral nucleic acid.
 9. The chimeric DNA of claim2, wherein the unpolyadenylated hairpin RNA lacks a 5′ cap structure.10. The chimeric DNA of claim 2, wherein the spacer sequence has alength of from 4 to 200 basepairs.
 11. The chimeric DNA of claim 2,wherein the promoter is a constitutive promoter.
 12. The chimeric DNA ofclaim 2, wherein the promoter is an inducible promoter.
 13. The chimericDNA of claim 2, wherein the promoter is recognized by a single subunitRNA polymerase from a bacteriophage.
 14. The chimeric DNA of claim 2,wherein the promoter is recognized by a eukaryotic RNA polymerase IIIand the DNA further comprises a terminator for the polymerase III. 15.The chimeric DNA of claim 2, wherein the target specific DNA regionwhich is transcribed to form the unpolyadenylated hairpin RNA lacks aDNA region involved in 3′ end formation and polyadenylation.
 16. Thechimeric DNA of claim 15, wherein the target specific DNA regioncomprises a 3′ end formation signal and lacks a polyadenylation signal.17. An animal cell in culture comprising a chimeric DNA which comprisesa promoter operably linked to a target specific DNA region which encodesan unpolyadenylated hairpin RNA, wherein the target specific DNA regioncomprises a target specific sense nucleotide sequence and a targetspecific antisense nucleotide sequence, wherein the target specificantisense nucleotide sequence comprises 20 consecutive nucleotides in asequence identical to the sequence of a complement of a part of an RNAmolecule transcribed or produced from a nucleic acid of interest in theanimal cell, wherein the target specific sense nucleotide sequencecomprises 20 consecutive nucleotides in a sequence identical to thesequence of the part of the RNA molecule transcribed or produced fromthe nucleic acid of interest, wherein the target specific sensenucleotide sequence and the target specific antisense nucleotidesequence are separated and linked by a spacer sequence, such that theunpolyadenylated hairpin RNA resulting from transcription of the targetspecific DNA region comprises the spacer sequence located between senseand antisense nucleotide sequences in the unpolyadenylated hairpin RNA,and wherein the sense and antisense nucleotide sequences arecomplementary to each other so as to form the unpolyadenylated hairpinRNA.
 18. The animal cell in culture of claim 17, wherein theunpolyadenylated RNA is expressed in the cell.